M.J. Gómez-Lechón

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP- β : LAP and LIP, HNF-3 α, HNF-4 α, RXR- α) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

Evaluation of the cytotoxicity of ten chemicals on human cultured hepatocytes: Predictability of human toxicity and comparison with rodent cell culture systems

The cytotoxic effect of the first 10 chemicals on the MEIC list (evaluated in the Multicentre Evaluation of In Vitro Cytotoxicity organized by the Scandinavian Society of Cell Toxicology) was evaluated on human and rat cultured hepatocytes and in the non-hepatic murine 3T3 cell line. The MTT test was used as an endpoint to evaluate cytotoxicity after 24 hr of exposure to the chemicals. The predictability of human toxicity using human hepatocytes was analysed and compared with the results using rodent cell culture systems and rat and mouse LD(50) tests. Ferrous sulphate, diazepam and isopropyl alcohol produced about the same toxicity in all three cell culture models; paracetamol and acetylsalicylic acid were more toxic to human and rat hepatocytes than to mouse 3T3 cells; amitriptyline, ethylene glycol, methanol and ethanol were more toxic to human hepatocytes than to rodent cells. Digoxin was the most cytotoxic chemical to human hepatocytes (IC(50), 4.9 nm), the alcoholic compounds (isopropanol, ethylene glycol, ethanol and methanol) were the least toxic (IC(50), 125-819 mm) and paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam and amitriptyline showed intermediate cytotoxicities (IC(50), 0.05-6 mm). The data suggest that for these 10 chemicals, acute toxicity in humans was more accurately predicted using human hepatocytes than using rat hepatocytes or mouse non-hepatic 3T3 cells.

Carbamazepine: a 'blind' assessment of CYP- associated metabolism and interactions in human liver-derived in vitro systems

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7- ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liverderived systems provide useful information for prediction of metabolic routes, rates and interactions.

Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell viability is the preculture of hepatocytes before cryopreservation. By the use of this procedure, high-quality cells, estimated in terms of post-thaw recovery, viability, adaptation of hepatocytes to culture, drug-metabolizing capability and cytochrome P450 induction, are obtained. Therefore, cryopreserved hepatocytes can provide a regular source of metabolically competent cells for in vitro investigations of the metabolic profile of new drugs and drug–drug interactions in pharmaco-toxicological research.

Semi-automatic quantitative RT-PCR to measure CYP induction by drugs in human hepatocytes

An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional liver tissue are incubated with the drugs for 50 h. Total RNA is extracted from culture and the cDNA is prepared by reverse transcription using high fidelity reverse transcriptase. Using appropriate primers, selective amplification of each CYP cDNA is achieved and real-time quantified by SYBR-Green fluorescence measurement. The extent of CYP induction obtained with the tested compounds is compared with the induction obtained with CYP model inducers (methylcholantrene, phenobarbital and rifampicin). This technique can be of value, to considerably simplify the identification of drug candidates with potential CYP inducing ability in man.

Enzyme-linked immunosorbent assay to quantify fibronectin

Fibronectin, a multifunctional soluble glycoprotein, is found in soluble form in blood and other body fluids. Fibronectin binds to biological substrata--collagen, gelatin, etc.--and to nonbiological substrata as well. An interesting finding was that fibronectin bound to polystyrene plastic quickly and tightly and further treatment of plastic with denaturing agents did not remove or denature it once it was adsorbed. Through the use of this property, a three-step noncompetitive enzyme-linked immunosorbent assay has been developed to quantify soluble fibronectin in biological samples. The following parameters were studied: selective attachment of fibronectin to polystyrene; interference caused by increasing amounts of external protein; and linear response and limit of sensitivity. The procedure has been applied to measure fibronectin from different animal sources.

Molecular mechanism of diclofenac hepatotoxicity: Association of cell injury with oxidative metabolism and decrease in ATP levels.

A certain number of case reports of adverse hepatic reactions to diclofenac are known, suggesting that diclofenac-associated hepatitis may be more common than previously recognized. In order to discriminate among possible molecular mechanisms of toxicity, the following were investigated: (a) cytotoxicity of diclofenac on metabolizing (rat hepatocytes) and non-metabolizing hepatic cells (HepG2, FaO); (b) changes in calcium homoeostasis, glutathione (GSH), lipid peroxidation and ATP levels, and (c) diclofenac metabolism in relation to cytotoxicity. The results indicate that toxicity is associated with the oxidative metabolism of the drug, and correlated with the formation of a minor oxidation metabolite. Inhibitors of diclofenac metabolism concomitantly reduced the toxicity of the drug. Hepatocyte injury was preceded by a decrease in ATP levels. No oxidative stress (no changes in GSH, no lipid peroxidation) could be demonstrated at this early stage. Cytotoxicity was prevented when cells were incubated with fructose, suggesting that the inability of mitochondria to produce ATP is the probable cause of diclofenac hepatotoxicity.

Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines : correlation between in vitro data and human lethal concentration

The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21-30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC(50) = 450-17,000 mum) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC(50) = 2-350 mum). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD(50)) only when low cytotoxic concentrations (IC(10)) were used for correlation.

Inhibition of monooxygenase activities in human hepatocytes by interferons.

The effects of human recombinant interferons alpha and gamma on monooxygenase activities in cultured human hepatocytes have been investigated. Dose-response and time course studies showed that interferons reduced 7-ethoxyresorufin O-deethylase activity of human hepatocytes after a 12-hr incubation with 300 U/ml interferons alpha and gamma (52% and 38% decrease, respectively). A reduction in 7-ethoxyresorufin O-deethylase activity was also observed in HepG2 cells, although in these cells maximal inhibition was observed after 24 hr of treatment with 1000 U/ml (a 41% and 28% decrease with interferon alpha and gamma, respectively). A decrease in activity was also observed in 7-pentoxyresorufin O-depentylase, 7-ethoxycoumarin O-deethylase and testosterone 2alpha- and 6beta-hydroxylase. It is noteworthy that the marked increase in 7-ethoxyresorufin O-deethylase activity detected in human hepatocytes after incubation with 2 mum-3-methylcholanthrene (10-fold over non-treated cells) was reduced by 40% in the presence of interferons (300 U/ml), thus indicating that the inducibility of monooxygenases could be altered by interferon treatment. The inhibitory effect of interferons on 7-ethoxyresorufin O-deethylase was transient and HepG2 cells recovered their normal activity 24 hr after interferon removal from culture medium. This study provides the first direct evidence that interferons down-regulate the level of monooxygenases in human hepatic cells and prevent, in part, their induction by xenobiotics.

Acute cytotoxicity of ten chemicals in human and rat cultured hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentrations

The cytotoxicity of ten chemicals from the MEIC list (nos 11-20) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the Multicentre Evaluation of In Vitro Cytotoxicity programme organized by the Scandinavian Society of Cell Toxicology. The lactate dehydrogenase intracellular activity and the MTT test were used as endpoints of cytotoxicity after 24 hr of exposure to the chemicals. Sodium chloride and lithium sulphate were the least cytotoxic compounds in all of the cellular systems (IC(50), 25-150 mm). The eight remaining chemicals (1,1,1-trichloroethane, phenol, sodium fluoride, malathion, 2,4-dichlorophenoxyacetic acid, xylene, nicotine and potassium cyanide) showed a similar cytotoxic potential in the four in vitro systems in a narrow range of concentrations (IC(50), 1-30 mm). The data suggest that these ten chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or to exert species-specific toxicity. The correlation between in vitro data and human lethal blood concentrations showed a relatively low predictability for the toxicity of six compounds with important lethal effects on the CNS. The predictability of the in vitro systems was similar to that of in vivo rodent tests (LD(50)) only when low cytotoxic concentrations (IC(10)) were used for the correlation.